Development of a Protective Cellular Therapy to Prevent Graft Versus Host Disease Post Allogenic Hematopoietic Stem Cell Transplant 2331939

Crosstalk Between Donor T Cells and Recipient IL-33+ Target Tissue Stroma Controls Alloimmune Responses Causing Graft-versus-Host Disease 2260079

Graft-versus-host disease (GVHD) remains a major complication of allogeneic hematopoietic cell transplantation (AlloHCT). Conditioning with chemotherapy and radiation enables donor engraftment but causes tissue injury that upregulates the alarmin interleukin-33 (IL-33) in fibroblastic reticular cells (FRCs) in secondary lymphoid organs (SLO) and tissue stromal cells. While mechanisms releasing IL-33 from nuclear sequestration remain elusive, free IL-33 directly activates donor CD4+ T cells to promote Th1 differentiation and expansion. Early IL-33 blockade limits GVHD initiation, yet the role of local IL-33 in sustaining alloimmunity remains unclear. We used preclinical models to define how IL-33 signaling to donor T cells within SLO versus the gastrointestinal tract (GIT) stroma controls active GVHD and graft-versus-lymphoma (GVL) responses. BALB/c recipients received B6 T cell—depleted bone marrow, A20 lymphomas, and T cells from donors with inducible ST2 (IL-33R) deletion to control IL-33 signaling during GVHD and GVL. To identify stromal functions, B6 recipients lacking IL-33 in FRCs (CCL19-Cre×Il33fl/fl) or gut epithelium (Vil-Cre×Il33fl/fl) were transplanted with BALB/c T cells and bone marrow. T cell—dependent IL-33 release was tested using GzmB⁻/⁻, St2+/+, and St2⁻/⁻ T cells. Late ST2 deletion resolved GVHD without compromising GVL. IL-33 signaling increased Foxp3 levels and reduced T-bet expression in donor CD4+ T cells, while sustaining TCF-1. Unexpectedly, loss of FRC-derived IL-33 exacerbated GVHD, whereas gut epithelial IL-33 deletion was protective. IL-33 induced GzmB, and GzmB⁻/⁻ T cells displayed impaired activation and expansion. Early SLO-derived IL-33 establishes effector programming that influences later tissue responses. IL-33+ stroma—T-cell crosstalk programs donor CD4+ T-cell fate and shapes local alloimmunity during GVHD. IL-33—stroma feedback is a key signal to alloreactive T cells during GVHD. NIH/NIAID F30AI147437, NIH T32 CA082084, NIH/NHLBI R01HL122489, NIH/NIAID R21AI121981, NIH/NIAID R56AI13927 Transplantation Immunology (TRAN)

[Novel therapeutic strategies to separate GVHD from GVL based on GVHD pathophysiology and mechanisms of leukemia relapse].

Lymphoid Tissue-Activated Donor T cells (LADTs) for Improved Donor Lymphocyte Infusion (DLI) against Blood Cancers 2250463

Donor lymphocyte infusion (DLI) is a key strategy for treating relapses after HLA-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT) for blood cancers. In DLI, T cell alloreactivity drives both the highly beneficial graft vs leukemia (GvL) effect and the potentially fatal graft vs host diseases (GvHDs), which severely limits DLI’s application and efficacy. Here, we propose to separate GvL and GvHD by engineering Lymphoid Tissue-Activated Donor T cells (LADTs) that display alloreactivity only in lymphoid tissues where cancer cells accumulate but not in organs involved in GvHD including the skin, liver and guts. To generate LADTs, PBMCs were first used to expand alloreactive T cells from an HLA-mismatched donor. Then, T cell alloreactivity was abrogated by knocking out CD3ε using CRISPR/Cas9, which blocks cell surface expression of the TCR/CD3 complex. Finally, the T cells were engrafted with a B cell-sensing mechanism that includes a CD19-specific synthetic Notch receptor (synNotch) and an expression cassette that drives the expression of an exogenous copy of CD3ε in response to synNotch engagement. This mechanism enables LADTs to restore alloreactivity only in lymphoid organs where B cells are abundant but not in other organs where B cells are scarce. We have established a process of generating LADTs with consistent yields. LADTs bearing CD19-specific synNotch receptors restored surface expression of TCR/CD3 in response to CD19+ cells and showed strong cytotoxicity against HLA+ U266 myeloma cells but not against HLA- ones. After removing CD19+ cells, LADTs gradually lost TCR/CD3 cell surface expression. Our study demonstrated the feasibility of LADT generation and the proof-of-concept of using LADTs for improved DLI. This cellular immunotherapy approach does not depend on cancer-specific biomarkers thus may be applied to a wide range of hematopoietic malignancies. Nemours Foundation Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

GATA3 expressing CD4 T cells promote intestinal graft-versus-host disease 2306818

Hematopoietic cell transplantation (HCT) is a curative therapy for blood cancers but is complicated by graft-versus-host disease (GVHD), where donor CD4+ T helper (Th) cells drive gut pathology. Defining factors that drive pathogenic Th cell differentiation in the gut promises to inform therapies that mitigate GVHD without impacting the beneficial anti-cancer activity of HCT therapy. In our work, we identified GATA3 as a key transcription factor that may facilitate the development of gut pathogenic Th cells after HCT. We hypothesized that Th cell deletion of GATA3 will impair pathogenic Th cell differentiation and improve clinical outcomes in a murine GVHD model. Using a murine allo-HCT model, we transplanted T cell-depleted bone marrow from WT C57BL/6 donors and splenocytes from C57BL/6-Gata3fl/fl Rosa26ERT2-Cre donors into irradiated BALB/c recipients. Donor cell GATA3 deletion was induced via tamoxifen treatment of recipient mice. Disease severity, intestinal pathology, donor cell trafficking, cytokine/granzyme expression, and innate immune infiltration were assessed by clinical scoring, histology, flow cytometry, and ex vivo Th cell stimulation. GATA3 deletion reduced intestinal GVHD pathology and impaired early donor T cell trafficking and persistence in the gut. While IFN-γ and IL-17A production were largely unaffected in early timepoints, GATA3 was required for heightened granzyme A expression, sustained GM-CSF production and chronic GM-CSF-driven intestinal eosinophilia at later timepoints. Analysis of human GVHD samples exhibited enrichment of eosinophil chemotactic pathways, mirroring murine findings. GATA3 promotes intestinal GVHD by supporting donor T cell gut infiltration, granzyme A expression, and sustained GM-CSF-dependent eosinophilia. These findings identify GATA3 as a key regulator of pathogenic Th responses and a potential therapeutic target for mitigating intestinal GVHD. Purdue Institute for Cancer Research Transplantation Immunology (TRAN)

CAR19 Tregs treat murine chronic graft-versus-host disease through immune suppression without measurable B cell cytolysis

Chronic graft-versus-host disease (cGVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic transplantation. cGVHD pathophysiology involves cooperation between T follicular helper cells (TFHs) and germinal center B cells (GCBs), allo- and autoantibody depositions in cGVHD tissues, and fibrosis. We evaluated human CD19–directed chimeric antigen receptor (CAR19) T cell therapy in a clinically relevant murine cGVHD model with bronchiolitis obliterans syndrome (BOS). Although CD8+ CAR19 T cells effectively reduced peripheral B cell and GCB frequencies, pulmonary function was unimproved. In contrast, a single infusion of CAR19 CD4+ regulatory T cells (Tregs) mitigated ongoing pulmonary disease and modulated germinal centers (GCs) associated with reduced TFH frequencies compared with control Tregs but without measurable B cell depletion. Compared with EGFR Treg infusion, mice receiving CAR19 Tregs exhibited enhanced suppression of B cell activation and preserved splenic architecture, and CAR19 Treg infusion provided greater opportunities for interaction with CD19+ B cells at the B cell follicle boundary zones. Taken together with the absence of detectable B cell cytolysis, these findings were most consistent with GC suppression rather than B cell depletion as the dominant mechanism. Overall, our findings suggest that CAR19 Tregs represent a promising and safe cGVHD/BOS therapeutic strategy, offering immunosuppressive benefits and improved disease outcomes that may be more limited with CD8+ CAR19 T cell treatment.

Timed STING Inhibition Mitigates Gastrointestinal GvHD While Preserving Graft-versus-Leukemia Activity After Allo-HSCT

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is curative for hematological malignancies but limited by graft-versus-host disease (GvHD), in which donor T cells damage host tissues. Current prophylaxis broadly suppresses donor immunity, often compromising the beneficial graft-versus-leukemia (GvL) response, highlighting the need for strategies that uncouple GvHD from GvL. The cGAS–STING pathway can be strongly activated during conditioning-induced tissue damage and represents a potential therapeutic target. However, its context-dependent roles in inflammation and homeostasis have constrained its clinical translation. Here, we show that “timed” administration of the covalent STING inhibitor H151 before conditioning reduced GvHD-associated mortality without impairing GvL in murine allo-BMT models. Donor T cell activation and effector function were preserved, indicating that the protective effect operates at the level of GvHD target tissues rather than through systemic immunosuppression. Timed STING inhibition protected the intestinal epithelium by limiting apoptosis, preserving intestinal stem cell function, and sustaining metabolic fitness during conditioning-induced injury, independently of type I interferon (IFN-I) signaling. In allo-HSCT patients, low intestinal STING expression is associated with reduced transplant-related mortality. Together, these findings identify timed STING inhibition as a tissue-protective prophylactic strategy that could be incorporated into existing conditioning regimens to enhance efficacy while minimizing toxicity.